The human auditory system has an exceptional ability to separate competing sounds, but the neural mechanisms that underlie this ability are not understood. Responses of inferior colliculus neurons to "mistuned" complex tones were measured to investigate possible neural mechanisms for spectral segregation. A mistuned tone is a harmonic complex tone in which the frequency of one component has been changed; that component may be heard as a separate sound source, suggesting that the mistuned tone engages the same mechanisms that contribute to the segregation of natural sounds. In this study, the harmonic tone consisted of 8 harmonics of 250 Hz; in the mistuned tone, the frequency of the 4th harmonic was increased by 12% (120 Hz). The mistuned tone elicited a stereotypical discharge pattern, consisting of peaks separated by approximately 8 msec and a response envelope modulated with a period of 100 msec, that bore little resemblance to the discharge pattern elicited by the harmonic tone or to the stimulus waveform. Similar discharge patterns were elicited from many neurons with a range of CFs, especially from neurons that exhibited short-latency sustained responses to pure tones. In contrast, transient and long-latency neurons usually did not exhibit the stereotypical discharge pattern. The discharge pattern was generally stable when stimulus level or component phase was varied; the major effect of these manipulations was to shift the phase of the response envelope. Simulation of IC responses with a computational model suggested that off-frequency inhibition could produce discharge patterns with these characteristics.